AFLP analysis and improved phytoextraction capacity of transgenic gshI-poplar clones (Populus canescens L.) in vitro

Clone stability and in vitro phytoextraction capacity of vegetative clones of R x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRI-MseI digestion. Four fragments generated by EcoAGT-MseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in R x canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO4 (10(-1) to 10(-5) m) incubated for 21 days on aseptic tissue culture media WPM containing 1 mu m Cu. Zn2+ caused phytotoxicity only at high concentrations (10(-1) to 10(- 2) m). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper

B801: Performance Evaluations of Potato Clones and Varieties in the Northeastern States 1983

Cooperative potato clone and variety trials were conducted at 23 locations to determine field, storage, and processing behavior of selected clones and varieties grown under soil, climatic, and cultural management common to the potato growing areas of 13 cooperating states and the Province of New Brunswick, Canada. These tests are all contributions to Regional Project NE107 entitled, Breeding and Evaluation of New Potato Clones for the Northeast. The primary objective of this project is to determine clone stability over a wide range of soil, climate, and cultural conditions.https://digitalcommons.library.umaine.edu/aes_bulletin/1119/thumbnail.jp

High-throughput screening for best clone manufacturing using scale-down models

The timelines for cell line development and early-stage process development are decreasing, but simultaneously there has been an increase in the amount of associated data. New methods in molecular and cell biology, new analytical methods, including the application of Process Analytical Techniques (PATs) and other methods to assess product quality early on, as well as new trends in process miniaturization and automation, increase the throughput in cell line and early-stage process development and the amount of data needed to be analyzed and reviewed. Thus, data structuring and curation represents a serious bottleneck to proper data analysis and valid decision making. We have designed a highly integrated data management and workflow system, which supports automated clone and manufacturing process development workflows, and provides the foundation to increase throughput in cell line and process development. The system is designed to continuously capture, in a highly structured manner, the heterogeneous data during the course of process development campaigns. It supports the whole process from expression, construct generation via transfection, cell seeding, selection, passaging, and cryo-conservation to cell culture and product quality analytics. This includes repeated passaging for clone stability assessments as well as (micro-) bioreactor runs for clone selection and process optimization. It can handle molecule, cell line, sample and process information as well as analytical test results. The system tracks the full history of all clones - from initial transfection all the way to their master cell banking and beyond - and provides lineage information for all samples handled during a campaign. Here, we present concrete use cases to demonstrate how the platform supports miniaturized screening approaches in scale-down models such as microtiter plate and micro-bioreactor based clone and process screening by capturing, processing, aggregating, and visualizing online and offline data and integrating them with product quality and molecule data to enable holistic process development for any type of biologics molecules (IgGs, novel antibody formats, novel scaffold, fusion proteins, enzymes, etc.)

Automated and enhanced clone screening using a fully automated microtiter plate-based system for suspension cell culture

In this talk we will present how we accelerated a clone selection process by 4 weeks while increasing the information density obtained for each clone. This was achieved by increasing the throughput and integrating new powerful analytical technologies as gene expressions and glycosylation analysis at a very early stage. Additionally, we could identify the overall top clone that was lost during the pre-selection phase of the reference process which is based on a considerably lower automation degree. Using our in-house developed MTP-based cell culture system for clone screening and selection offers substantial potential for improvement and acceleration: The system provides a fully automated process and enables fed-batch cultivation at an early stage which is of high importance for successful clone selection. Due the high throughput of more than 600 cultivations at the same time the MTP-based system considerably increases the number of clones that can be evaluated and speeds up the processing. The potential to integrate real-time monitoring of metabolites and other secondary selection criteria crucial for productivity and product quality further supports reliable clone selection. Moreover, the MTP-based system establishes an interface for advanced HT analytics to identify additional parameters for clone evaluation. Thus, we demonstrated the technical feasibility to couple the MTP-based cell culture system with glycosylation analysis as well as the LightCycler® technology to perform automated RT-qPCR gene expression analysis for a large number of cell culture samples. The results of the RT-qPCR analysis showed that the identified top clone displayed the highest mRNA expression level for the HC of the examined mAb which correlated with the highest specific productivity recorded at the protein level. Furthermore, RT-qPCR analysis may also be beneficial to monitor the expression of stress markers like chaperones and factors related to endoplasmic reticulum stress potentially correlating with product concentration or it can be applied to improve prediction of clone stability attributes related to promoter methylation or transgene copy number. This particular advancement combined with the high flexibility of the system opens up future perspectives for optimizing the selection process at an early stage, e.g. by using multiple selection criteria that are especially tailored for each product. The early availability of product quality data will also improve the chances to select the most suitable clone and to reduce risks and required effort during later development stages. This is of particular importance when expressing complex molecule formats such as bispecific antibodies, glycoengineered antibodies or antibody cytokine fusion proteins. Our high-throughput MTP-based cell culture system appears most suitable to enhance efficiency and robustness of the clone screening procedure as well as the quality of the selected clones. Product quality control can be reached by selecting the clone with the desired product quality pattern at a very early stage using our cell culture system that is proven to be predictive for large scale bioreactors

Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones <it>via </it>recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, <it>oriV</it>, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate <it>trans</it>-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer <it>oriV</it>-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a <it>P</it>_BAD-driven <it>trfA </it>gene generating strain MW005 that supports, independently, both recombineering and copy-number induction.</p> <p>Results</p> <p>The <it>P</it>_BAD-driven copy of <it>cre </it>in EL350 was replaced seamlessly with a copy of <it>trfA</it>, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based <it>Caenorhabditis elegans </it>genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation.</p> <p>Conclusions</p> <p>By retrofitting EL350, an established 'recombineering' <it>E. coli </it>strain, with a tightly regulated copy of <it>trfA </it>we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of <it>oriV</it>-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.</p

Production of a new anti-A monoclonal reagent

Monoclonal antibodies are essential tools in molecular and cellular immunology research. They have essentially replaced the polyclonal antibodies in identifying blood groups and detecting cell markers and pathogenic agents. The aim of the present study is to produce monoclonal antibody identifying the ABO blood groups using the murine hybridoma technology. An anti-A monoclonal antibody A907 was selected and estimated for its use in the manufacture of a reagent anti-A. The selected antibody specifically reacts with A1, A2/sub>, A1B and A2B erythrocytes. It does not recognize B, O, A3 and Ax erythrocytes. The A907 monoclonal antibody can be used in blood grouping in association with a reagent recognizing the A weak phenotypes.Key words: Anti-A monoclonal antibodies, murine myeloma cells, haemagglutination.African Journal of Biotechnology Vol. 4 (8), pp. 844-84